Synthesis, Antimicrobial, and Antioxidant Activities of Chalcogen-Containing Nitrone Derivatives from (R)-citronellal

Background: The main constituents of Cymbopogonnardus (L) Rendle and C. citratus (DC) Stapfessential oils are (R)-citronellal and citral, respectively. Organochalcogen compounds can boost the biological activities of natural products. Methods: Several chalcogen-containing nitrones derived from (R)-citronellal and citral were prepared and evaluated for their antimicrobial and antioxidant activities. The antimicrobial activity was evaluated by the disc diffusion test and the antioxidant properties were evaluated in vitro by DPPH (1,1-diphenyl-2-picryl-hydrazyl), ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), and FRAP (ferric ion reducing antioxidant power) assays. Results: In the antimicrobial assay, (E)-N,3,7-trimethyl-3-(phenylthio)oct-6-en-1-imine oxide 5c exhibited halos between 21.5 mm (Escherichia coli O157:H7) and 26.0 mm (Listeria monocytogenes), while (E)-N,3,7-trimethyloct-6-en-1-imine oxide 5d presented halos between 22.5 mm (E. coli O157:H7) and 31.0 mm (L. monocytogenes). (E)-N,3,7-Trimethyl-2-(phenylthio)oct-6-en-1-imine oxide 5a showed the lowest minimal inhibitory concentration (MIC) value against Bacillus cereus (0.48 mM), and 5c was the most potent bactericide, with a minimal bactericidal concentration (MBC) of 0.52 mM for E. coli O157:H7. In the antioxidant assays, 5c, 5d, and 10 ((E)-3,7-dimethyl-2-(phenylselanyl)oct-6-enal oxime) were the most actives in the DPPH, ABTS, and FRAP assays, respectively. Conclusions: The presence of a phenylthio group in the nitrone increases its antimicrobial activity against Gram-positive and Gram-negative foodborne pathogens in the disk diffusion test and the antioxidant activity in vitro.


General procedure for the synthesis of nitrones 5a-d derived from citronellal
Using a synthetic route adapted from Isager et al. [3] in a 25 mL vial was added the aldehyde 1, 3a-b or 8 (0.5 mmol), N-methyl-hydroxylamine hydrochloride (4, 0.084 g, 1 mmol) and water (2 mL) as the solvent and the mixture was stirred at room temperature for 30 min. Then, a 1M solution of Na2CO3 (1.0 ml) was added and the stirring was continued for additional 24 h. Compound 5a was purified by preparative chromatographic plate (silicagel) and compounds 5b-d were isolated by column chromatography using neutral alumina as a stationary phase and a solution of hexanes/ethyl acetate as the eluent (90:10). The NMR spectra of nitrones 5a ( Figure S1 and Figure S2), 5b ( Figure S3 and Figure S4), 5c ( Figure S5 and Figure S6) and 5d ( Figure S7 and Figure S8) are in accordance with those expected for the compounds.

Synthesis of the selenium-containing oxime 10
Using a synthetic route adapted from Isager et al. [3], in a 25 mL vial was added α-phenylseleno citronellal (5b, 0.156 g, 0.5 mmol), hydroxylamine hydrochloride (9, 0.069 g, 1 mmol) and water (2 mL) as the solvent. After stirring for 30 min at room temperature, it was added 0.5 mL of an aqueous solution of Na2CO3 (0.027 g, 0.26 mmol) and the stirring was continued for additional 22 h. After this time, the oxime 10 was isolated by column chromatography using silicagel as a stationary phase and a solution of hexanes/ethyl acetate (95:5) as the eluent. The NMR spectra of oxime 10 ( Figure S9 and Figure S10) are in accordance with those expected for the compound.

Antimicrobial activity assay using the disk diffusion test
The disk diffusion test followed the methodology recommended by the Clinical Laboratory Standards Institute CLSI [4]. The inoculum was standardized by standard McFarland to the concentration of 10 8 CFU.mL −1 and spread on the surface of a Petri dish containing Mueller-Hinton (MH) agar. Then, the paper disc (6 mm) was put on a plate impregnated with 20 μL of the testing compound previously diluted in DMSO (1:1) and incubated at 37 °C for 24 hours. Zone of inhibition ≥20 mm were considered as strong inhibition, <20-12 mm as moderate inhibition, and <12 mm no inhibition. A positive control with streptomycin antibiotic (10 μg) and a negative one, with paper discs impregnated with distilled water, were used

Determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
The MIC was determined using the macro dilution tube method, in accordance with Rota et al. [5], with modifications. The tested concentrations were 20, 18, 15, 12, 10, 7 and 5 μL.mL -1 of each compound. In a tube with 1 mL of MH broth, was inoculated 10 5 UFC.mL -1 , the compound in a predetermined concentration and, to facilitate the solubility, 2 drops of TWEEN 80. The mixture was incubated at 37 °C for 24 h under stirring (150 rpm). As the positive control, MH broth and inoculum concentration in 10 5 UFC.mL -1 and as the negative control MH, the inoculum concentration in 10 5 UFC•mL -1 and streptomycin (10 μg) were used. MIC was defined as the lowest concentration of compound which showed no visible growth in broth. After 24 h, tubes with no visible growth were seeded in a 100 mL TSA-YE tube and incubated at 37 °C for 24 h. The MBC is the lowest concentration of compound where 99.9% of the initially inoculated cells were killed.

Antioxidant activity assays
The antioxidant properties of the synthesized compounds were evaluated by three different methods in vitro: DPPH and ABTS + radical scavenging activity and ferric ion reducing antioxidant power (FRAP). All drugs were dissolved in dimethyl sulfoxide (DMSO). The experimental results were given as the means ± standard deviation (SD) to show the variations among the groups. The statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Newman-Keuls multiple comparison test when appropriate. The differences were considered statistically significant at a probability of less than 5% (p < 0.05). All tests were performed at least three times in duplicate. The IC50 values (the concentration of sample required to scavenge 50% of the free radicals) were calculated from the graph of the scavenging effect percentage versus the compound concentration.

Radical Scavenging Activity
To determine if compounds 5a-d and 10 present in vitro antioxidant activity against free radicals, DPPH and ABTS + scavenging capability were evaluated at different concentrations.
The DPPH scavenging activity of compounds 5a-d and 10 (10-500 μM) was determined in accordance with the method of Sharma and Bhat [6] and the decrease in the absorbance at 517 nm was recorded.
The ABTS radical scavenging activity was determined according to the method described by Erel [7]. Different concentrations of compounds 5a-d and 10 (10-500 μM) were mixed with the ABTS + solution, and the decrease in the absorbance at 734 nm was recorded.
The values are expressed as the percentages of radical inhibition (I %) in relation to the control values, as calculated by the following equation: (1) Where Ac is the absorbance of the control excluding the test compounds, and As is the absorbance of the tested compounds.

Ferric ion reducing antioxidant power (FRAP)
The ferric ions (Fe 3+ ) reducing antioxidant power (FRAP) method was used to measure the reducing capacity of compounds 5a-d and 10. The assay was performed as described by Stratil et al. [8] with slight modifications . Different concentrations of 5a-d and 10 (1-500 μM) and FRAP reagent were added to each sample, and the mixture was incubated at 37 °C for 40 min in the dark. The absorbance of the resulting solution was measured at 593 nm with a spectrophotometer.

In vitro toxicity
The activity of -ALA-D in the presence of compounds 5a-d and 10 at different concentrations (10-500 μM) was determined according to the method described by Sassa [9]. Tissues were removed from the mice, liver and kidneys (1:10 w/v) and brain (1:5 w/v). They were homogenized in a 0.1 M Tris-HCl buffer, pH 7.4 and centrifuged at 2500 RPM for 10 min. The supernatants were used for determination of the -ALAD activity.
Firstly, 200 μL of the tissue supernatant was incubated for 10 min at 37 °C with 50 μL of potassium phosphate buffer (TFK), 150 μL of distilled water, and 10 μL of the test compound. Then, 100 μL of substrate (12 mM ALA aminolevulinic acid) was added and the incubation was carried out at 37 °C for 1 h for liver and kidney and 3 h for the brain; the reaction was stopped by adding 250 μL of trichloroacetic acid (TCA) solution with 10% 10 mM HgCl2 followed by centrifugation (2500 RPM for 10 min). Then, 500 μL of the supernatant was collected and added to 1000 μL of Ehrlich's reagent and 500 μL of distilled water. The resulting mixture was incubated for 10 min at room temperature and the absorbance was read at 555 nm.
The results were expressed in nmol PBG.mg.tn Where T is the incubation time, ptn is protein and tissue refers to the added amount, for example 0.